This invention relates to an autofocus technique and more particularly to an autofocus methodology for use with fluorescence scanners.
In laser fluorescence scanning it is critical to eliminate as much as possible fluorescence (and scattering) from materials other than the surface-bound nucleic acids which have been tagged with fluorescing molecules. One method for reducing such background from sources outside the detection plane is the use of depth discrimination. In such a system, signals outside a detection plane are substantially minimized. The system, however, must make sure to keep the detection plane at the peak of the sensitivity vs. depth curve. If the system fails to keep the detection plane at the peak of the sensitivity vs. depth curve there will be a non-uniformity of the scanned images. Thus, in order to improve performance in fluorescence scanning, a need exists for focusing with good precision onto the plane that has a fixed (preferably zero) distance from the surface of the substrate being scanned.
In one aspect, the invention is an autofocus apparatus including a laser for generating a collimated laser beam. A lens is positioned to receive the laser beam off-axis and to focus the beam at a fixed focal plane at a substrate surface. A first position sensitive detector receives the laser beam reflected from the substrate surface through the lens to generate a first signal proportional to lateral beam offset. A beam splitter may direct a portion of the laser beam before passing through the lens toward a second position sensitive detector to generate a second signal proportional (except for possible offsets) to laser beam pointing errors. In this case, additional apparatus may be used to compute the difference of the positions from the first and second position sensitive detectors, which will be (except for possible offsets) proportional to the defocus error signal corrected for laser pointing error. As will be appreciated by those skilled in the art, this defocus error signal can be used in a control loop to reduce this error signal to achieve autofocus. In one embodiment, an offset is added so that the system will focus on a plane that differs from the substrate surface. It is preferred that the first position sensitive detector and the second position sensitive detector be located approximately the same total distance from the laser.